This in vitro study compared the detrimental effect and relative biological effectiveness (RBE) of high-linear energy transfer (LET) fast neutrons on rat immature hippocampal cultured cells with those of low-LET γ rays. Immature hippocampal cells were exposed to fast neutrons or γ rays. Cytotoxicity and cell viability were analyzed using a lactate dehydrogenase (LDH)-release assay and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, respectively. The cytotoxicity and cell viability with fast neutrons or γ rays varied in a dose-dependent pattern. In the LDH release and MTT assay indices, the RBEs of fast neutrons were approximately 2.35 and 2.42, respectively. Fast neutrons markedly induced apoptotic changes in immature hippocampal cells with increased expression of active caspase-3 and cleaved poly(ADP-ribose) polymerase. Increased cytotoxicity and decreased cell viability in immature hippocampal cells were seen in a dose-dependent pattern after fast-neutron and γ irradiation. Fast neutrons have a higher RBE for cell death indices than γ rays.